As part of a large, ongoing study of invasive infections in pediatric patients in Bamako Mali, 106 cases of invasive pneumococcal disease (IPD) were identified from June 2002 to July 2003. Of the 12 serotypes present, the majority of isolates were not contained in the PCV7 conjugate vaccine, including 1 isolate that was serotype 1, 12 serotype 2, 58 serotype 5, 7 serotype 7F, and 1 serotype 12F. To determine if clonal dissemination of the predominant serotypes had taken place, genotyping was performed on 100 S. pneumoniae isolates using two methods: pulsed-field gel electrophoresis (PFGE) of SmaI-digested genomic DNA, and the Bacterial Barcodes rep-PCR method using the Enterococcus kit. Criteria for delineating rep-PCR genotypes were established such that isolates of different serotypes were generally not grouped together. The two methods were equally discriminatory within a given pneumococcal serotype. PFGE separated the isolates into 15 genotypes and 7 subtypes; rep-PCR separated isolates into 15 genotypes and 6 subtypes. Using either method, isolates within serotypes 2, 5, and 7 formed three large, separate clusters containing 1 genotype each. Both methods further distinguished related subtypes within serotypes 2 and 5. One of the PFGE subtypes of serotype 5 is indistinguishable from the Columbia5-19 clone circulating in Latin America since 1994. The data suggest that the predominance of serotypes 2 and 5 is likely the result of dissemination of clones, some of which are responsible for invasive diseases over a broad population range. These results have recently been submitted for publication.